Immunogenicity of A Novel Live Attenuated Tuberculosis Vaccine. Summary. Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (M. tb), is a global problem that currently affects approximately one-third of the world?s population and results in nearly 1.4 million deaths each year. The current tuberculosis vaccine, M. bovis BCG (BCG), has variable protection levels ranging from 0-80%. Earlier research from our group has identified several M. tb mutants that were further developed into live attenuated vaccine (LAV) candidates against TB. Using a stringent aerosol model of murine challenge, immunization with these candidates (M. tb?mosR, M. tb?echA7) significantly reduced the M. tb load in murine tissues to undetectable levels for one of the constructs with the production of robust cell mediated immunity (CMI). More importantly, it protected mice against challenge with the Beijing clade of M. tb isolates where the current BCG vaccine shows insufficient protection. However, the long-term protective responses of these vaccines have not been determined. The overarching hypothesis of this project is that mosR-based vaccines will induce a broader and more potent memory T-cell response, will be more effective against highly virulent M. tb clinical isolates, and will be able to counteract the activity of regulatory T cells, three areas in which BCG performs very poorly. In this project, we will test our hypothesis and dissect the protective immune responses for this promising TB vaccine candidate by; A) Evaluating the safety and stability of MTBLAV (M. tb?mosR?echA7). We will construct a double knock mutant dubbed here as MTBLAV (M. tb?mosR?echA7) and assay its stability on both genomics and transcriptome levels. In addition, the safety of MTBLAV will be assayed in both immune-competent (C57BL/6) and immune- compromised (Rag-/-, IFN?-/-) mice (Aim I). In the Second Aim of the project; B) We will dissect the generation and maintenance of immune responses induced by MTBLAV in mice. We will analyze the development of long-term vaccine immunity and T-cell responses elicited by the MTBLAV (?mosR?echA7) compared to BCG using the murine model of TB following aerosol challenge with virulent clinical isolate of M. tb Beijing 4619. By completing these aims, we will better characterize and understand the ability of this novel vaccine to protect against tuberculosis. In future project, we will further dissect the generated immunity in other TB models, more relevant to human TB (such as non-human primates).